We have characterized a novel isoform of nonmuscle myosin II-C (NM II-C), NM II-C2, which is generated by alternative splicing of an exon, C2, encoding 41 amino acids in mice (33 in humans). The 41 amino acids are inserted into loop 2 of the NM II-C heavy chain within the actin binding region. Unlike most vertebrate nonmuscle and smooth muscle myosin IIs, baculovirus expressed mouse heavy meromyosin (HMM) II-C2 demonstrates no requirement for regulatory myosin light chain (MLC20) phosphorylation for maximum actin-activated MgATPase activity or maximum in vitro motility as measured by the sliding actin filament assay. In contrast, noninserted HMM II-C0 and another alternatively spliced isoform HMM II-C1 which contains 8 amino acids inserted into loop 1 are dependent on MLC20 phosphorylation for both actin-activated MgATPase activity and in vitro motility (Kim, K. Y., Kovacs, M., Kawamoto, S., Sellers, J. R., and Adelstein, R. S. (2005) J. Biol. Chem. 280, 22769-22775). HMM II-C1C2, which contains both the C1 and C2 inserts, does not require MLC20 phosphorylation for full activity similar to HMM II-C2. These constitutively active, C2-inserted isoforms of NM II-C are expressed only in neuronal tissue. This is in contrast to NM II-C1 and NM II-C0, both of which are ubiquitously expressed. Full length NM II-C2-GFP expressed in COS-7 cells localizes to filaments in interphase cells and to the cytokinetic ring in dividing cells.